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Definitions

• the present invention relates generally to encapsulated vaccines and methods of making same, and more particularly, to oral vaccines that release an embedded bioactive agent at the site of action along the foregut and hindgut of an animal and the vaccine is embedded in a cross-linked matrix that is protected by a glassy matrix of sugars.
• Polymer microspheres and lamellar particles can be employed for mucosal administration of antigens. Because vaccines themselves may not be efficiently recognized and taken up by mucosal lymphocytes, they typically need to be coadministered with penetration enhancers or adjuvants.
• Different classes of polymer mixtures are known for potential use as mucoadhesives (Malik, Baboota et al. 2007). These include synthetic polymers such as poly (acrylic acid) (PAA), hydroxypropyl methylcellulose and poly-(methylacrylate) derivatives, as well as naturally occurring polymers such as hyaluronic acid and chitosan.
• Chitosan has been extensively used for a variety of applications as a biomaterial for tissue engineering, wound healing, and as an excipient for drug delivery (Chopra, Mahdi et al. 2006; Dang and Leong 2006). Chitosan has occasionally been tested as an adjuvant for mucosal application (Kim, Kim et al. 2007), but it is typically applied directly to a mucosal surface such as intranasal application in order to obtain IgA response in the nasopharyngeal mucosa of terrestrial animals (Kang, Jiang et al. 2007). Chitosan has also been shown to possess useful properties such as non-toxicity, high biocompatibility and non-antigenicity.
• Chitosan can be obtained through the deacetylation of chitin, the major compound of exoskeletons in crustaceans.
• Chitosan [a-(l ⁇ 4)-2-amino-2-deoxy- ⁇ -D-glucan], a mucopolysaccharide closely related to cellulose, exhibits chemical properties that are determined by the molecular weight, degree of deacetylation, and viscosity.
• Chitosan can form microparticles and nanoparticles that can encapsulate large amounts of antigens (van der Lubben, Verhoef et al. 2001; Davis 2006). In the acidic environment of the stomach, chitosan retains its positive charges that hold the particle together.
• Chitin and chitosan are being extensively used in the pharmaceutical industry (cosmetics, contact lenses, artificial skin, wound dressing), paper making, photography, solid state batteries, waste water treatment, chromatography, dietary supplements and animal feed. Processing techniques for the preparation of chitosan microspheres have been extensively developed since the 1980s. Several processing approaches have been proposed including ionotropic gelation with an oppositely charged, simple or complex coacervation, emulsif ⁇ cation/solvent evaporation and, more recently, spray drying (Huang et al. 2003). Chitosan microspheres obtained by spray drying are characterized by high sphericity and specific surface area, which are important parameters for application in the pharmaceutical field (Rege, 2003).
• TPP sodium tripolyphosphate
• the present invention provides a composition and a method for the manufacturing of a mucoadhesive delivery system for the oral vaccination of animals.
• the mucoadhesive delivery vehicle releases the vaccine at the site of action (i.e., the Gut Associated Lymphoid Tissue; GALT) along the foregut and hindgut of the animal.
• the mucoadhesive delivery vehicle is incorporated in the regular food or beverage normally consumed by the animal.
• the mucoadhesive delivery vehicle is provided in a form of dry or semi-dry particles comprising an immunogenic substance (i.e., the vaccine), which is embedded in a composite matrix of cross-linked mucoadhesive polymer and protected by sugars.
• an immunogenic substance i.e., the vaccine
• the present inventors have found a way to produce large quantities of mucoadhesive particles containing vaccines with minimal drying efforts under sterile conditions and while using only food grade ingredients.
• the particles when incorporated in a food product and administrated orally, the particles elicited both mucosal and systemic immune responses.
• compositions and a method of preparing a particle comprising a bioactive agent that is embedded in one or more mucoadhesive polymers, the one or more mucoadhesive polymer being further embedded in a glassy matrix, wherein the glassy matrix comprises: 10-60 wt % of sugars, 3-10 wt % of a oligosaccharides and 1-10 wt % of electrolytes, wherein the electrolytes, such as di- or poly-valent anion or cation compounds, act as cross-linking agents.
• the present invention also provides a process of preparing a particle encapsulating a bioactive agent, the method comprising the steps of dissolving one or more mucoadhesive polymers in aqueous solution, admixing the bioactive agent under ambient temperature and slightly acidic conditions, extruding the slurry into a counter ion solution to form firm hydrogel beads or strings and saturating the hydrogel particles with sugars.
• the sugar saturated hydrogel particles are further dehydrated by desiccation to reduce the moisture content to below 20%.
• the semi-dry particle materials are subjected to further drying and milling to obtain microparticulated powder containing the bioactive agent and having moisture content below 10%.
• the present invention provides a semi-wet particle as described above, where the hydrogel particles are chopped for further size reduction without further drying.
• the present invention provides a bioactive agent embedded in a mucoadhesive polymeric matrix and is further stabilized and thermo-protected by glassy matrix of sugars.
• the present invention provides a process of preparing a particle containing a bioactive agent where the hydrogel particles are saturated with sugars which significantly reduces the amount of free water in the particles and the associated need for extensive drying step.
• the present invention provides a process of preparing a particle containing a bioactive agent where all ingredients including salts and solutions are food grade ingredients, non-toxic, biodegradable and naturally occurring ingredients.
• Yet another aspect of the present invention provides an immunogenic particle comprising a bioactive agent, one or more mucoadhesive polymer and an emulsif ⁇ er all of which are cross-linked with a phosphate containing agent to form a hydrogel particle, and wherein the hydrogel particle is embedded in a sugar matrix.
• the present invention has a number of unexpected advantages over the prior art. Thanks to the specific formulation and process conditions, the water content in the particles is very minimal while at the same time the formula also stabilizes the sensitive bioactive. A further important advantage is the excellent thermo-stability of the encapsulated ingredient. Without being bound by theory, it is believed that the amorphous sugar glassy structure created around the bioactive agent provides protection against the deleterious effects of heat and oxidation. This permits the particles of the present invention to be incorporated in a food product, the preparation of which entails pressure, sheer force, and heat treatments. Further advantages include the free-flowing characteristics of the particles of the invention as well as ability to control the particle size over a wide range from 5 microns to over 5000 micron.
• Figure 1 shows a schematic side view of the apparatus for producing the particles of the current invention.
• Figure 2 shows the hydrogel strings before (upper picture) and after (bottom picture) harvesting from vessel B.
• Figure 3 shows the optimum treatment conditions for immunogen release from the particles.
• Bioactive Agent refers to naturally occurring, synthetic, or semi-synthetic materials (e.g., compounds, fermentates, extracts, cellular structures) capable of eliciting, directly or indirectly, one or more physical, chemical, and/or biological effects.
• the bioactive agent may be capable of preventing, alleviating, treating, and/or curing abnormal and/or pathological conditions of a living body, such as by destroying a parasitic organism, or by limiting the effect of a disease or abnormality.
• the bioactive agent may further be referred to as a pharmaceutical agent (such as an immunogen, a prophylactic agent, a therapeutic agent), a diagnostic agent, and/or a cosmetic agent, and includes, without limitation, vaccines, prodrugs, affinity molecules, synthetic organic molecules, polymers, low molecular weight molecules, proteinaceous compounds, peptides, vitamins, steroids, steroid analogs, lipids, nucleic acids, carbohydrates, precursors thereof, and derivatives thereof.
• the bioactive agent may also be a nutritional supplement.
• Non-limiting nutritional supplements include proteins, carbohydrates, water-soluble vitamins (e.g., vitamin C, B-complex vitamins, and the like), fat-soluble vitamins (e.g., vitamins A, D, E, K, and the like), and herbal extracts.
• the active agent may be commercially available and/or prepared by known techniques.
• Microencapsulation is defined as a process that produces a composition containing a bioactive agent that is in the form of a microparticle in the size range of 10 to 5000 ⁇ m, or a composition that can be milled to a microparticle in the size range of 10 to 5000 ⁇ m.
• Complex is defined as interaction between two molecules or portions of the same molecule through noncovalent interactions such as coordination bonds, electrostatic interactions, hydrogen bonding interactions, and hydrophobic interactions.
• Particle refers to a particulate that is solid (including substantially solid or semisolid, but excluding gel, liquid and gas), having an average geometric particle size (sometimes referred to as diameter) of less than 5 mm, preferably 500 microns or less, more preferably between 100 microns and 5 microns. Particles may be formed from, in part or in whole, with one or more non-limiting materials, such as the bioactive agents, mucoadhesive polymers, carriers, polymers, stabilizing agents, and/or complexing agents disclosed herein.
• Immunogenic substances are defined as a bioactive, a substance or a composition of matter, which is capable of mounting a specific immune response in an animal.
• Immunogenic substances would include immunogenic peptides and proteins including mixtures comprising immunogenic peptides and/or proteins (e.g., bacterins); intact inactive, attenuated, and infectious viral particles; intact killed, attenuated, and infectious prokaryotes; intact killed, attenuated, and infectious protozoans including any life cycle stage thereof, and intact killed, attenuated, and infectious multicellular pathogens, recombinant subunit vaccines, and recombinant vectors to deliver and express genes encoding Immunogenic proteins (e.g., DNA vaccines).
• immunogenic proteins e.g., DNA vaccines
• Vaccination is defined as a process that results in a specific immune response generated by an animal against an immunogen or an immunogenic substance.
• a "Muco adhesive Delivery System” or “Mucodhesive Delivery Vehicle” is defined as a composition that results in the delivery of bioactive agent, an immunogen or an immunogenic substance to the desired location in the intestinal or nasal mucosa.
• “Mucoadhesive Polymer” refers to a natural, synthetic, or semi-synthetic molecule having two or more repeating monomer units in a main chain or ring structure. Polymers broadly include dimers, trimers, tetramers, oligomers, higher molecular weight polymers, substituted derivatives thereof, and mixtures thereof. The polymer may be ionic or non- ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
• a "Mucoadhesive" molecule is a component of a mucoadhesive delivery system that specifically binds to mucosal tissues.
• Such molecules include, but are not limited to chitosan, alginate, hyloronic acid and cationic guar.
• Amorphous refers to the glassy state of materials and constructions that lacking crystallinity or otherwise non-crystalline.
• a "Glassy Matrix” for the purpose of the present invention is an amorphous solid characterized by viscosities and an extremely low molecular mobility. The presence of a glassy state can be confirmed by establishing characteristic differential scanning calorimetry curves, for which particles are generally brought to the rubbery state by slow and continuous heating of the material.
• "Semi-Dry or Semi- Wet” refers to a state that includes at least substantially solid and/or semi-solid, but excludes gel, liquid, and gas.
• Cross-linking generally refer to the linking of two or more materials and/or substances, including any of those disclosed herein, through one or more covalent and/or non-covalent (e.g., ionic) associations.
• Cross-linking may be effected naturally (e.g., disulfide bonds of cystine residues) or through synthetic or semi-synthetic routes. As described herein, cross-linking refer to converting polymeric slurry into a firm structure of hydrogel particles using salts having counter ions.
• Oral administration of vaccines offers several advantages. Dosages can be administered to a large number of animals via the food or water with minimal labor and stress to the animal. Adverse immune reactions following oral administration are also much less likely to occur. For meat producing animals, oral administration has the additional advantage of avoiding common reactions or infections at injection site, broken needles, or the use of highly reactive adjuvant. These reactions decrease the value of the animal at harvest.
• An effective oral delivery system requires a delicate balance among factors such as the simplicity of preparation, cost effectiveness, high loading level of the bioactive agent, controlled release ability, storage stability, and effective immunogenicity of the components.
• the method and process described herein offers significant advantages compared to other particulate delivery systems, including the conventional micro- and nano-encapsulation systems. It is also expected that the problems of instability, low loading level, and cost effectiveness are better resolved with the polymeric mucoadhesive system of the current invention.
• the present invention provides a composition and a method for manufacturing of mucoadhesive particles containing one or more bioactive agents.
• bioactive agents may be microencapsulated in accordance with this invention, the invention will be described below primarily with reference to the microencapsulation of Immunogenic Substances that are bacterin vaccines.
• the present invention enables the oral delivery of an immunogenic vaccine useful in the prevention of disease in animals including aquatic animals.
• a mucoadhesive polymer such as but not limited to chitosan, at a concentration of 1-10% (w/w), is dispersed in 1-5 N acetic acid solution at a temperature in the range of 2O 0 C to 65 0 C until fully dissolved.
• Indigestible short chain oligosaccharide components may be added to improve protection of the antigen from stomach acidity, bile acids, and proteases, and to increase the intestinal adsorption of the bioactive agent. Examples of materials that could be used include, but are not limited to, inulin and fructooligosaccharides (FOS).
• the pH of the slurry is then brought to about 5.8 and 0.5-10% (w/w) of a natural emulsif ⁇ er such as lecithin is added to form a stable emulsion.
• a natural emulsif ⁇ er such as lecithin
• One or more mono or disaccharides are then added to achieve a saturated emulsion with a concentration of from 5-50% (w/w).
• the emulsion is stabilized by the interaction between positive charge of the mucoadhesive polysaccharide, the emulsif ⁇ er, and hydroxyl groups of the sugars and oligosaccharides.
• the increased hydrophobicity and elasticity of the mucoadhesive polysaccharide and emulsif ⁇ er helps delay or prevent penetration of water or gastric juices into the matrix once formed into particles.
• the sugars and phospholipid complex increases the stability of the bioactive agent and protects the agent against heat.
• the added sugars dramatically reduce the free water in the slurry, allowing for faster dehydration and drying.
• the pH of the slurry is then gradually increased to about pH 6.2 by the addition of base and a solution containing the bioactive agent added.
• Cross-linking is used to promote the formation of stable hydrogel particles.
• Various cross-linking agents have been used in fixing polymeric gels. These cross-linking agents are mostly synthetic chemicals such as formaldehyde, glutaraldehyde, dialdehyde, starch, glyceraldehydes, cyanamide, diimides, dimethyl adipimidate, diisocyanates, and epoxy compounds.
• these chemicals are all highly cytotoxic which will restrict their utility in food applications.
• glutaraldehyde is known to have allergenic properties and is cytotoxic at concentrations greater than 10-25 ppm and as low as 3 ppm in tissue culture.
• a cross-linking agent suitable for use in food applications that is within acceptable cytotoxicity and that forms stable and biocompatible cross-linked products.
• a food grade product having a Generally Recognized As Safe (GRAS) status as a cross-linking agent (sodium triphosphate) has been used to form stable hydrogel particles.
• the resulting solution/suspension is then dropped or extruded into a cross-linking solution containing water-soluble phosphate salts.
• a salt exchange reaction (cross-linking) takes place, resulting in the formation of hydrogel beads or linear threads in which the bioactive agent is retained.
• the resulting suspension of particles containing the embedded bioactive agent is then soaked in a sugar saturated solution, collected, and dewatered to form semi-dry particles that can be further dried by a number of means well known in the art such as freeze drying, vacuum drying, spray drying, and the like, and milled to produce fine powder having a size range of from 5 to 5000 microns. Details of the manufacturing process are set out in the series of steps described below:
• a solution comprising a bioactive agent such as, but not limited to, an immunogen or immunogenic substance(s) is dissolved into the slurry described above prior to cross- linking.
• the resulting composition is then allowed to fall in drops, or in a continuous stream, into a cross-linking solution of 1-10% sodium triphosphate solution.
• the slurry can be spray-atomized into an aqueous solution containing 1-10% sodium triphosphate.
• a chitosan/tripolyphosphate molecular mass ratio of at least about 4:1 is maintained.
• the wet particles beads or threads are harvested from the cross-linking bath by any suitable means well known in the art (e.g., screening, filtration, centrifugation, and the like) and soaked in a saturated sugar solution followed by mixing in any food acceptable desiccation compounds such as silica gel, starch granules and the like for further dewatering.
• a saturated sugar solution followed by mixing in any food acceptable desiccation compounds such as silica gel, starch granules and the like for further dewatering.
• the sodium triphosphate and the sugar saturated solutions can be reused for cross-linking more batches of chitosan slurry.
• the silica gel can also be washed, sterilized, and reused to dewater additional batches.
• the semi-dry particles are mixed in the feed formula for subsequent pelleting or extrusion.
• the semi-dry particles can be further dried using conventional processes well known in the art such as, but not limited to, freeze drying, vacuum drying, fluidized bed drying and tunnel drying.
• the dried material is then milled and sieved to the appropriate particle size class if necessary.
• the final sized particles can be mixed directly with the feed materials for subsequent pelleting or extrusion, or it can be mixed with edible oil for top-coating of a standard commercially available feed for oral administration.
• Figure 1 demonstrates the process equipment which includes an airtight, stainless steel steam jacketed insulated mixing vessel (A) provided with a high-speed homogenizer from 250 - 20,000 RPM.
• the temperature in vessel-A is controlled from about 20 0 C to 80 0 C.
• the mucoadhesive slurry containing the bioactive agent is prepared in vessel-A as described above. Nitrogen gas pressure from about 5-25 psi is then applied and controlled by Pressure regulator-1 to force the slurry through Feed line 2 ( Figure 1).
• the process tank (Vessel-B) is where the mucoadhesive polymer hydrogel beads or threads are formed.
• the airtight all stainless steel vessel is equipped with a liquid jet unit connected to Feed line-2, an exhaust system (Exhaust f ⁇ lter-3), and a removable stainless mesh basket (Mesh basket-4) that is raised about 10 cm from the vessel bottom.
• the liquid height in vessel-B is sufficient to maintain a minimum of 80 cm distance between the jet and the surface of the cross-linking solution in the tank which is necessary for allowing the stream of beads or continuous threads to settle at the bottom while hardening, thereby clearing the solution surface to accept freshly introduced material.
• the density of the slurry can be further adjusted by the addition of insoluble salts such as, but not limited, to calcium carbonate or calcium sulfate. This will allow for a rapid sinking of the beads or continuous threads to the tank bottom, thereby clearing the solution surface for freshly introduced material. Otherwise material may accumulate at the surface and prevent effective cross-linking of freshly introduced particles or threads.
• FIG. 2 shows hydrogel strings before (upper picture) and after (bottom picture) harvesting from vessel B.
• Vessel-C is similar to Vessel-B and serves for sugar soaking and dewatering of the hydrogel particles. Initially the tank filled with a solution containing at least 40% sugars or, more preferably, a sugar saturated solution. The hydrogel particles or threads are soaked in the sugar solution under vacuum of from about 1-100 mBARS for 30-60 min then allowed to drip dry by raising the mesh basket as described above. The sugar solution is removed from the tank and a thick layer of food compatible desiccating agent such as, but not limited to, silica gel beads or starch granules, is introduced to vessel-B. The mesh basket containing the hydrogel particles or threads is placed on top of the desiccating agent layer in vessel-C.
• desiccating agent such as, but not limited to, silica gel beads or starch granules
• a vacuum pressure from aboutl-100 mBARS is applied again for additional 120 min.
• the semi-dry material containing 10-20% moisture is then removed from the vessel and chopped to smaller size and directly incorporated in a feed formula for pelleting or extrusion.
• the semi-dry material can be further dried to a moisture content of below about 10%, followed by milling and sieving through mesh screens of an appropriate size (e.g., from about 50-500 micron).
• the sodium triphosphate solution in Vessel-B and sugar saturated solution in Vessel-C can be reused for cross-linking more batches of chitosan slurry, and the silica gel in Vessel-C washed, sterilized, and reused to dewater additional batches.
• the diameter of beads or strings produced by this method will vary depending on the jet diameter and stream velocity.
• the jet diameter found to be useful in producing the particles was in the range from 50 to 7000 microns.
• the vertical jet velocity found to be useful was in the range of 0.1 cm 3 /sec to 10 cm 3 /sec.
• One of the advantages of the present invention is in the controlling of jet velocity by the nitrogen gas pressure applied in Vessel-A and the simple sterilization procedure which effectively eliminates the cumbersome use of a typical pumping system.
• the particles are removed from the mesh basket and directly placed in a drying unit without the dewatering step in Vessel-C. Due to the sugars saturation step, the hydrogel particles contain only about 30-40% water and thus require a minimal drying process to further reduce the moisture content to below 10%.
• the slurry is transferred from Vessel-A under a pressure of nitrogen gas to a separate pressure vessel connected to Feed line-2, and Vessel-C is used for sugar saturation while a separate similar Vessel-D is used for desiccation.
• Vessel-C is used for sugar saturation while a separate similar Vessel-D is used for desiccation.
• Example 1 Production of mucoadhesive polymer particles
• 180 L of sterile distilled water is added and warmed to 5O 0 C.
• Three L of glacial acetic acid is carefully added (with mixing) to 17 liters of distilled water in an open flame hood.
• the diluted acetic acid solution is then slowly added to the distilled water in the steam jacketed stainless steel tank.
• Chitosan (4 kg high viscosity chitosan, Sigma, St. Louis, MO) is slowly added to the warmed acetic acid solution under a vigorous homogenizing (10,000 RPM) until completely dissolved.
• the chitosan solution is cooled to room temperature and the pH adjusted to 6.2 with 50% sodium hydroxide solution.
• Liquid soy lecithin (6 kg, Archer- Daniels-Midland Co., Decatur, IL) is added to the chitosan solution and the solution emulsified for 15 minutes under vigorous homogenization (10,000 RPM).
• a solution of bacterin containing 5 million injectable doses is added to the chitosan slurry mixture prepared above.
• a pressure of 15 psi is then applied to the vessel and the slurry forced through 1 A quarter of inch feed line connected to a liquid jet head unit located at the center top cover of a 1000 L airtight steam jacketed stainless steel flat bottom vessel.
• the vessel is equipped with #10-12-mesh stainless steel basket located about 10 cm above the vessel bottom.
• the vessel contains 300 L of 10% w/w sodium triphosphate solution.
• the chitosan slurry is forced through a 5 mm liquid jet head unit that produces a uniform stream of the slurry into the sodium triphosphate solution.
• Uniform size strings of hydrogel are instantly formed and sink to the bottom of the meshed basket.
• the hydrogel strings are allowed to harden for 2 hours in the sodium triphosphate solution. After fully hardened, the meshed basket is raised above the solution surface and allowed to drip for an additional hour.
• the mesh basket containing the hydrogel string material is then transferred to a similar 1000 L airtight stainless steel flat bottom vessel containing about 100 liter of sucrose saturated solution and a vacuum of 10 mBARS is applied for 30 minutes facilitating the absorption of the sugars by the hydrogel particles.
• the mesh basket is then raised above solution surface and the material is allowed to drip dry for 2 hours.
• the mesh basket containing the sugar saturated hydrogel material is then transferred to another similar 1000 litter airtight stainless steel flat bottom vessel containing about 100 kg of #35-60 mesh food grade and sterile silica gel.
• the vacuum on the tank is reduced to about 10 mBARS for 1 hour to provide further dewatering.
• the semi-dry hydrogel material containing about 20% moisture is chopped to small pieces of less than 1 mm in size for incorporation in a feed formula for pelleting or extrusion.
• the semi-dry hydrogel material can be further dehydrated to below 10% moisture in a vacuum drier or fluidized bed drier.
• Example 2 Production of mucoadhesive polymer particles using high fructose corn syrup (HFCS).
• HFCS high fructose corn syrup
• Hydrogel Particles or strings are prepared as in Example 1.
• Sterile HFCS solution (Archer-Daniels-Midland Co., Decatur, IL) containing 55% fructose is used to saturate the hydrogel particles.
• 100 L of HFCS solution is added to a 1000 L airtight stainless steel flat bottom vessel and hydrogel particles are allowed to soak in the HFCS for about 30 minutes as described above in example 1.
• the particles are further dewatered in silica gel and semi-dry hydrogel material containing 20% moisture is obtained following the dehydration procedure described in example 1.
• the semi-dry hydrogel material is further dried in a fluidized bed drier at 50 0 C to achieve a moisture level less than 10%.
• the resulting dried material is then milled using an industrial hammer mill and the powder is sieved to less than 100-microm-particle size.
• Example 3 Production of mucoadhesive polymer particles containing Salmonid Rickettsial Septicaemia (SRS) vaccine.
• SRS Salmonid Rickettsial Septicaemia
• the mucoadhesive polymer slurry at pH 6.2 was prepared as described in Example 1.
• a solution containing attenuated SRS vaccine (5 x 10 17 /ml SRS bacterin) (commercially available from Centrovet, Santiago, Chile) was mixed with yeast extract immunostimulator (500 g beta glucan, AHD International, Atlanta, GA) and added into the slurry.
• yeast extract immunostimulator 500 g beta glucan, AHD International, Atlanta, GA
• the hydrogel material was allowed to harden for 2 hour and then saturated with sucrose. It was then further dewatered in silica gel, freeze dried, and milled to particle size lower than 100 micron.
• ELISA Enzyme-Linked ImmunoSorbant Assay
• ELIS A's were typical sandwich style in which the microtiter plates were coated with primary sheep antibody P. salmonis (Sigma).
• the SRS antibodies were captured overnight at 4°C, and the plate was reacted with a secondary anti-sheep IgG (Sigma HRP conjugate), followed by the anti-sheep alkaline phosphatase conjugate (Jackson Immunoresearch, West Grove, Pa.).
• the alkaline phosphatase is detected with para-nitrophenyl phosphate and read at 405 nm on a SpectroMax plate reader (Molecular Devices, Sunnyvale, CA).
• Example 4 Production of Bioadhesive particles with enhanced immunogenic properties.
• the slurry is then injected into 10% w/v sodium triphosphate solution.
• the hydrogel material is allowed to harden for 2 hour and then saturated with sucrose.
• the hydrogel is further dewatered in Sharpies centrifuge and dried in a freeze dryer.
• the resulting dried material is then milled using an industrial hammer mill and the powder is sieved to less than 100-microm-particle size.
• Example 5 Production of Bioadhesive particles containing a viral antigen vaccine against viral infections in fish.
• composition of the present invention is also effective in oral vaccination against viral infections such as infectious salmon anemia virus (ISAV), infectious pancreatic necrosis virus (IPNV), salmon swimbladder sarcoma virus (SSSV), etc.
• HIV infectious salmon anemia virus
• IPNV infectious pancreatic necrosis virus
• SSSV salmon swimbladder sarcoma virus
• One injectable dose may be equivalent to 2-20 micro liter solution of attenuated virus vaccine or yeast lysate containing recombinant proteins obtained from sequences of isolated virus. Both types of vaccines are available commercially from Centrovet, Santiago, Chile or Novartis animal Health, Greensboro, NC. USA.
• a typical formulation involves the preparation of slurry as described in Example 1.
• a solution containing 5 million doses of attenuated virus or proteins recombinants are mixed with yeast extract immunostimulator (500 g beta glucan, AHD International, Atlanta, GA) and added into the slurry.
• yeast extract immunostimulator 500 g beta glucan, AHD International, Atlanta, GA
• the slurry is then injected into 10% w/v sodium triphosphate solution.
• the hydrogel material is allowed to harden for 2 hour and then saturated with sucrose. It is then further dewatered in silica gel or Sharpies centrifuge, freeze dried, vacuum dried or fluidized bed dried, and milled to particle size lower than 100 micron.
• Example 6 Production of Bioadhesive particles containing a viral antigen vaccine against ISAV.
• yeast lysate solution (equivalent to 5 million injectable doses) containing proteins recombinants obtained from sequences of isolated virus of a Chilean origin (available commercially from Centrovet, Santiago, Chile) is mixed with yeast extract immunostimulator (500 g beta glucan, AHD International, Atlanta, GA) and added into a slurry as described in example 1.
• yeast extract immunostimulator 500 g beta glucan, AHD International, Atlanta, GA
• the hydrogel material is allowed to harden for 2 hour and then saturated with sucrose.
• the hydrogel is further dewatered in Sharpies centrifuge and dried in a freeze dryer. The dried material is milled to particle size lower than 100 micron.
• Example 7 Production of Bioadhesive particles containing a viral antigen vaccine against IPNV.
• VLP virus-like particle
• yeast extract immunostimulator 500 g beta glucan, AHD International, Atlanta, GA
• yeast extract immunostimulator 500 g beta glucan, AHD International, Atlanta, GA
• the slurry is then injected into 10% w/v sodium triphosphate solution.
• the hydrogel material is allowed to harden for 2 hour and then saturated with sucrose.
• the hydrogel is further dewatered in Sharpies centrifuge and dried in a freeze dryer.
• the resulting dried material is then milled using an industrial hammer mill and the powder is sieved to less than 100- microm-particle size.
• Example 8 Production of Storage Stable Bioadhesive particles containing a viral antigen vaccine against viral infections in fish.
• the final drying step of the composition of the present invention may be carried out in a way that allowed the formation of a sugar glass matrix surrounded the vaccine particles.
• a glassy formation stabilizes and protects the vaccine under unfavorable storage conditions of high temperature and humidity.
• Oral bacterin vaccines against a bacterial disease such as an SRS or viral vaccines against virus infections such as ISAV or IPNV were prepared as described in Example 6 or 7 and the final drying step of the sugar loaded thin threads or strings were dried in such a way that a sugar glass is formed.
• the thin threads were loaded on a 13X10" tray (13 x 10 inch) at a loading capacity of 800 g/sq ft and placed in a freeze drier (Virtis Advantage, Virtis, Gardiner, NY).
• the Condenser is chilled to -50 0 C, shelf temperature was adjusted to 40° degree C and the material allowed to warm up to about 35 0 C (measured by a pair of temperature sensors plugged in the wet material). Vacuum was then initiated and controlled at about 2500 mTORR through an external vacuum controller, (Thyr-Cont, Electronic, GmbH). As vacuum pulled down the product temperature fall and stabilized at about ⁇ 2°C. After 16 hours, the product temperature had increased to about +10 0 C. At this point, a full vacuum pressure was applied and shelf temperature rose to 50 0 C. Twelve hours after establishing full vacuum pressure, the dried product was taken out of the freeze drier and milled to particle size lower than 100 micron.
• Example 9 Production of Atlantic salmon feed containing SRS immunogenic microparticles
• Example 10 Production of mucoadhesive polymer particles containing Mycoplasma hyopneumoniae (M. hyo) vaccine.
• Mycoplasma hyopneumoniae (M hyo) is a widespread pathogen, found in swine herds throughout the world. The organism is associated with the development of bronchopneumonia. The disease leads to severe productivity losses by way of reduced weight gain and poor food conversion rates in the growing pigs.
• the slurry (pH 6.2) was prepared as described in Example 2. A solution containing 5 million doses of attenuated M. hyo vaccine commercially available from Boehringer Ingelheim Limited, Berkshire UK. is mixed with 50O g yeast extract immunostimulator (pure beta glucan, AHD International, Atlanta, GA) and added into the slurry.
• Example 11 Oral vaccination of pigs against M. hyo using the immunogenic microparticles of the present invention.
• Example 12 Nasal vaccination of pigs against M. hyo using the immunogenic microparticles of the present invention.
• the semi-dry hydrogel particles produced as described in examples 2 and 10 are dried in a vacuum drier to further reduce the moisture content to below 10%.
• the dry material is milled using an industrial hammer mill and sieved to particle size less than 30 micron.
• Growing pigs are immunized with the immunogenic microparticles via the respiratory tract and growth performances of vaccinated and non- vaccinated pigs recorded.

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